Methods for the Treatment of Fibromyalgia and Chronic Fatigue Syndrome

ABSTRACT

The invention relates to methods for the treatment of fibromyalgia and chronic fatigue syndrome by administration of a transdermally applied androgen composition. The treatment is both safe and effective for treating fibromyalgia-related pain and fatigue, as well as chronic fatigue syndrome.

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/837,310, filed Jul. 15, 2010, which application is acontinuation of U.S. patent application Ser. No. 11/303,813, filed Dec.16, 2005, now U.S. Pat. No. 7,799,769 B2, which application is acontinuation of U.S. patent application Ser. No. 10/464,310 filed Jun.18, 2003, abandoned. Each of these applications is incorporated hereinby reference in its entirety.

INTRODUCTION

The invention relates to the treatment of fibromyalgia and chronicfatigue syndrome by administration of an androgen composition. Thetreatment is effective for treating fibromyalgia-related pain andfatigue.

BACKGROUND OF THE INVENTION

The Women's Health Initiative (WHI) clinical trial, whose aim was toprospectively evaluate the risks and benefits of orally administeredcombination hormone replacement therapy in healthy women using estrogensand medroxyprogesterone acetate, was recently halted (Fletcher, S. W. etal. 2002. J. Amer. Med. Assoc. 288:366-368). The increased risks incoronary heart disease, breast cancer, stroke, and pulmonary embolismoutweighed the increased benefits in colorectal cancer, endometrialcancer, hip fractures and death due to other causes, resulting in asmall but statistically significant increased risk for the global indexof hazard ratios among women taking these hormones. The authors pointedout, however, that their study only evaluated healthy women, not thosewith symptoms of hormone deficiency. Furthermore, other routes ofdelivery, e.g., transdermal systems, need to be studied, since it ispossible that transdermal delivery may increase benefits and/or decreaserisks to these patients. It was noted by the authors of the WHI studythat hormone replacement therapy is still considered to be effective forrelieving perimenopausal symptoms such as hot flashes.

Most clinical trials evaluating sex hormone replacement therapy havefocused on estrogens and progestins, although testosterone replacementtherapy in women who may be testosterone deficient is now beginning tobe addressed using transdermal delivery systems, e.g., for diseasestates in which there is stress from chronic disease with loss of musclemass and chronic fatigue, such as wasting syndrome in women with AIDS(Miller, K. et al., 1998. J. Clin. Endocrinol. Metab. 83:2717-2725;Javanbakht, M. et al., 2000. J. Clin. Endocrinol. Metab. 85:2395-2401).Testosterone replacement therapy using transdermal delivery has alsobeen of benefit to men with symptoms of testosterone deficiency, forexample in men with Parkinson's disease (Okun, M. S. et al., 2002. Arch.Neurol. 59:1750-1753). There is accumulating evidence that the sexhormones, in particular estrogens, progestins and now testosterone, areimportant for subjective feelings of well-being and quality of life,parameters that were not assessed in the Women's Health Initiativetrial.

U.S. Pat. No. 5,935,949 discloses a method of alleviating the symptomsof fibromyalgia syndrome and chronic fatigue syndrome which involvesoral administration of androgens, such as testosterone, to patients. Theidea behind the use of testosterone therapy in the treatment of suchconditions is that muscle pain and chronic fatigue, primary symptoms inwomen with fibromyalgia syndrome (FMS), relates, at least in part, totestosterone deficiency, since androgens are known to allow forincreased musculature and improvement in fatigue. Indeed, a smalldecrease in serum free testosterone concentrations has been documentedfor premenopausal fibromyalgia patients relative to healthy volunteers,but significance was not achieved for postmenopausal women (Dessein, P.H. et al., 1999. Pain 83:313-319). A relationship between testosteroneand pain sensation has been previously suggested (Blomqvist, A. 2000.Compar. Neurol. 423:549-551). Accumulating evidence supports the conceptthat sex hormones can elevate the pain threshold in an individual, forexample, during pregnancy (Gintzler, A. R. 1980. Science 210:193-195),when testosterone concentrations, as well as estrogen and progesteroneconcentrations, are elevated (Bammann, B. L. et al., 1980. Am. J.Obstet. Gynecol. 137:293-298). The theory that testosterone can suppresspain is supported by the discovery of aromatase-positive cells in thespinal cord dorsal horn of higher vertebrates (quail), where initialprocessing of pain sensation occurs (Evrard, H. et al., 2000. J. Compar.Neurol. 423:552-564). The presence of aromatase, which convertstestosterone to 17.beta.-estradiol, is interesting because it is knownthat estrogen can induce the transcription of opiates in estrogenreceptor-positive cells derived from the superficial layers of thespinal dorsal horn (Amandusson, A. et al., 1996. Neurosci. Lett.196:25-28; Amandusson, A. et al., 1996. Eur. J. Neurosci. 8:2440-2445;Amandusson, A. et al., 1999. Pain 83:243-248), a location that isimportant for the synthesis of endogenous opiates. Administration ofestrogen to ovariectomized female rats has been demonstrated to increasespinal cord enkephalin transcription (Amandusson, A. et al., 1999. Pain83:243-248), and estrogen receptor-positive cells co-localize withpreproenkephalin mRNA (Amandusson, A. et al., 1996. Eur. J. Neurosci.8:2440-2445). These endogenous opiates act on enkephalinergic neurons tomediate inhibition of nociceptive relay cells, both in primary afferentfibers as well as in pain-modulating fibers descending from thebrainstem (Ma, W. et al., 1997. Neuroscience 77:793-811). Thus, bothtestosterone and estrogen appear to be important for modulating thesensation of fibromyalgia-related pain. However, the differentialimportance of androgens versus estrogens in pain sensation relative togender remains poorly understood.

Testosterone may also act at the level of the brain. Testosteroneconcentrations were dramatically decreased in the brain and spinal cordof rats in response to pain-inducing subcutaneous injections of formalininto the paw. In these animals, the loss of testosterone in the centralnervous system was demonstrated to be due to its metabolism by5.alpha.-reductase to dihydrotestosterone (Amini, H. et al., 2002.Pharmacol. Biochem. Behav. 74:199-204). These authors pointed out thatdihydrotestosterone can be metabolized to5.alpha.-androstane-3α,17β.-diol, which is an effective modulator ofGABA_(A) receptor complexes in the brain. GABA_(A) receptors are foundthroughout the brain, and actions of GABA_(A) receptor modulators in thelimbic system, specifically in the amygdala, are associated withfeelings of fear. The GABA_(A) receptor ion channel complex is one ofthe most important inhibitory ion channels in the brain. Thus,testosterone may be important not only for modulation offibromyalgia-related pain but also for feelings of emotional well-beingvia binding of its metabolites to the neurosteroid site of the GABA_(A)receptor, although this remains to be demonstrated.

Other hormones such as growth hormone may also play a role in thepathogenesis and symptoms of fibromyalgia and chronic fatigue. Forexample, studies have shown that fibromyalgia patients fail to exhibit aproper growth hormone response to acute exercise, a response that islikely related to increased levels of somatostatin a powerful inhibitorof growth hormone synthesis (Crofford, L. J. et al., 2002. Arthr.Rheumat. 46:1136-1138; Paiva, E. S. et al., 2002. Arthr. Rheumat.46:1344-1350). It is well known that testosterone increases growthhormone secretion. Growth hormone secretion is reduced in senescencebeyond the reduced levels of secretion seen in adult life after puberty.This reduction is thought to relate to the decreased lean body mass toadipose mass ratio known to occur in some individuals in senescence.Thus, increased somatostatin levels may reflect decreased anabolism anddecreased muscle mass due to decreased testosterone and growth hormoneconcentrations in fibromyalgia patients. As a result, therapy withgrowth hormone may improve the condition of patients with fibromyalgia.

It has now been found that transdermal hormone therapy in women cansafely and effectively raise serum hormone concentrations to levels thatapproximate those normally found in premenopausal women, as well asrelieve symptoms in patients with fibromyalgia.

SUMMARY OF THE INVENTION

An object of the present invention is a composition for increasingandrogen levels in blood which comprises an androgen at a concentrationof about one percent and a pharmaceutically acceptable gel. The androgencompounds of the instant invention may comprise testosterone and itsderivatives.

Another object of the present invention is a method of alleviating thesymptoms of fibromyalgia syndrome and chronic fatigue syndrome whichcomprises administering to a patient suffering from fibromyalgiasyndrome or chronic fatigue syndrome an effective amount of the androgengel formulation so that the symptoms are alleviated.

The invention relates to a method of alleviating the symptoms of acondition which is associated with deficient serum androgen levels in afemale human patient comprising transdermally administering daily tosaid patient suffering from deficient serum androgen levels a safe andeffective amount of an androgen which is both effective for alleviatingthe female patient's condition associated with androgen deficiency andfor consistently raising the female patient's serum androgen levels tothe middle-upper female reference range female reference range, whereinthe composition contains a daily unit dose of an androgen and isformulated to provide steady state total androgen serum levels withoutraising free androgen serum levels or twenty-four hour free androgen AUCabove the levels required for both therapeutic efficacy and safety.

The invention also relates to a method of determining the appropriateandrogen dosage for a female human subject comprising diagnosing thefemale human subject as having fibromyalgia or chronic fatigue syndromeaccompanied by a free androgen level in the lower half of theappropriate reference range; starting transdermal androgen treatment ofthe subject based on a diagnosis of fibromyalgia or chronic fatiguesyndrome. After a predetermined time of treatment the free androgenlevel in the subject's blood is measured. If the subject's free androgenlevel remains in the lower half of the appropriate reference range, theandrogen dosage is increased; if the subject's free androgen level iswithin the middle-to-upper end of the appropriate range, the androgendosage is maintained; or if the subject's free androgen level is inexcess of at least about 15% of the appropriate reference range, theandrogen dosage is decreased. The predetermined treatment time can be 30days, 60 days, 4 weeks, 8 weeks or any other time desired by a physiciantreating the subject. The method can be repeated several time over thecourse of treatment

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict the levels of total testosterone in blood of thepatients. FIG. 1A shows the day 1 profile, FIG. 1B shows the day 28profile, and FIG. 1C shows the means ±SEM for day 1 (open symbols)versus day 28 (filled symbols).

FIGS. 2A-2C depict the levels of free testosterone in blood of thepatients. FIG. 2A shows the day 1 profile. FIG. 2B shows the day 28profile. FIG. 2C shows the means ±SEM for day 1 (open symbols) versusday 28 (filled symbols), with a change in the y-axis scale.

FIG. 3 depicts the results of the tender point evaluations pre-treatment(day 0) and at the end of the study (day 28). The results reported arelevels of fibromyalgia-related pain on a scale of 0 (no pain) to 10(highest level of pain).

FIG. 4 depicts the results of the dolorimetry assessment of tender pointfibromyalgia-related pain pre-treatment (day 0) and at the end of thestudy (day 28).

FIGS. 5A-5B depicts the severity of symptoms/conditions associated withfibromyalgia and chronic fatigue on a scale of 1 to 10 (10 being thehighest increased level) on day 1 versus day 28 of the study. Thesymptoms/conditions assessed included libido, fibromyalgia-relatedmuscle pain, tiredness, headache severity, headache frequency,stiffness, sleeplessness, fatigue upon awakening, anxiety, anddepression.

FIGS. 6A-6M show the total and free testosterone levels for each of theindividual patients and a graph showing the mean of the subjects and thevalues for C_(min), C_(max), and C_(ave)

FIG. 7 is an example of a Fibromyalgia Study, Preliminary PatientQuestionnaire.

FIG. 8 is an example of a Physician Questionnaire Eligibility CriteriaForm.

FIG. 9 is an example of a Dose (or Study) Coordinator Serum TestosteroneValues Form.

FIG. 10 is an example of a Physician Evaluation Form (Tender Points).

FIG. 11 is an example of a Physician General Health Form.

FIGS. 12A-12D show an example of a Patient Questionnaire Form.

FIGS. 13A-13F show an example of a Global Health Form.

FIGS. 14A-14B show an example of a Fibromyalgia Movement Test Form.

FIG. 15 is an example of Patient Instructions.

FIG. 16 is an example of a Patient Log for Taking Study Gel.

FIG. 17 is an example of a Patient Checklist for Exercising.

FIG. 18 is an example of an Adverse Experience Report.

DETAILED DESCRIPTION OF THE INVENTION

The syndrome of chronic fatigue has received much attention lately. Nophysical finding or laboratory test can be used to confirm diagnosis ofchronic fatigue syndrome. However, this syndrome is generallycharacterized by fatigue persisting or relapsing for more than sixmonths occurring concurrently with at least four or more of thefollowing symptoms: impaired memory or concentration, sore throat,tender cervical or axillary lymph nodes, fibromyalgia-related musclepain, fibromyalgia-related multi-joint pain, new headaches, unrefreshingsleep, and post exertion malaise. Early studies suggested an infectiousor immune dysregulation mechanism for the pathophysiology of chronicfatigue syndrome. More recent studies have shown that neurologic,affective and cognitive symptoms also frequently occur.

Fibromyalgia (also referred to as fibrositis) is one of the most commonrheumatic syndromes in ambulatory general medicine affecting 3-10% ofthe general population. Most patients with Fibromyalgia Syndrome (FMS)are women, and of these patients, approximately 50-75% are women intheir peri-postmenopausal years, aged 40-60. Approximately 2-5% ofperi/post menopausal women are affected by FMS, with some estimatesranging from 0.5 to 20%. This disease is characterized by chronicwidespread fibromyalgia-related musculoskeletal pain syndrome withmultiple tender points, fatigue, headaches, lack of restorative sleepand numbness. Fibromyalgia shares many features with chronic fatiguesyndrome including an increased frequency in peri/post menopausal woman,absence of objective findings and absence of diagnostic laboratorytests. Further, these conditions have overlapping clinical featuresincluding chronic fatigue, headaches and lack of restorative sleep withmusculoskeletal fibromyalgia-related predominating in fibromyalgia andapparent increased susceptibility or hyperimmunologic responsiveness toinfection predominating in chronic fatigue syndrome.

Various treatments for chronic fatigue syndrome including acyclovir,oral and vaginal nystatin and fluoxetine have been tried with littlesuccess. Placebo-controlled trials have demonstrated modest efficacy ofamitriptyline, fluoxetine, chlorpromazine, or cyclobenzaprine intreating fibromyalgia. Exercise programs have also been suggested asbeneficial in both conditions. Accordingly, there is clearly a need forbetter treatments for these debilitating conditions.

It has now been found that transdermal administration of hormones,including androgens, can alleviate symptoms in patients suffering fromFMS or CFS. By “androgen therapy” it is meant to include administrationof a single androgen or a combination of androgens. By “alleviate” it ismeant to make less hard to bear, reduce or decrease, or lighten orrelieve patients of the symptoms of FMS of CFS. By “symptoms” of FMS orCFS it is meant to include fibromyalgia-related muscle pain and atrophy,chronic fatigue, lack of restorative sleep, increased susceptibility toinfection and headaches resulting from FMS or CFS.

The invention relates to a method of alleviating the symptoms of acondition which is associated with deficient serum androgen levels in afemale human patient comprising transdermally administering daily tosaid patient suffering from deficient serum androgen levels a safe andeffective amount of an androgen which is both effective for alleviatingthe female patient's condition associated with androgen deficiency andfor consistently raising the female patient's serum androgen levels tothe middle-to-upper range of the female reference range, wherein thecomposition contains a daily unit dose of an androgen and is formulatedto provide steady state total androgen serum levels without raising freeandrogen serum levels or twenty-four hour free androgen AUC above thelevels required for both therapeutic efficacy and safety.

The invention also relates to a method of determining the appropriateandrogen dosage for a female human subject comprising diagnosing thefemale human subject as having fibromyalgia or chronic fatigue syndromeaccompanied by a free androgen level in the lower half of theappropriate reference range; starting transdermal androgen treatment ofthe subject based on a diagnosis of fibromyalgia or chronic fatiguesyndrome. After a predetermined time of treatment, for example 15-30days, the free androgen level in the subject's blood is measured. If thesubject's free androgen level remains in the lower half of theappropriate reference range, the androgen dosage is increased; if thesubject's free androgen level is within the middle-to-upper end of theappropriate range, the androgen dosage is maintained; or if thesubject's free androgen level is in excess of the standard error of themean above appropriate reference range, the androgen dosage isdecreased. The predetermined treatment time can be from 1-6 weeks,determined by a physician treating the subject. The predetermined timecan be 30 days, 40 days, 4 weeks, or any time frame determined by thephysician. The method can be repeated several times over the course oftreatment, so that the appropriate dose of the androgen can be adjustedto avoid side effects.

Most trials involving hormone replacement therapy have used derivativesof hormones naturally found in women. These derivatized hormones havebeen promoted because of their patentability and their extended halflife. Androgens are no exception since the androgen hormone mostprescribed for women is methyltestosterone, where methylation at theC-17 position increases its oral bioavailability. Patients do nottolerate these derivatized hormones very well, however. Non-derivatizedexogenous hormones that are structurally identical to endogenoushormones have short plasma/serum half lives that range from 10-100minutes, making oral administration of native hormones problematic.Investigators have begun to develop transdermal delivery systems, whichprovide sustained delivery while minimizing hepatotoxicity. Atestosterone skin patch has been effective in HIV seropositive womenwith wasting syndrome (Miller, K. et al., 1998. J. Clin. Endocrinol.Metab. 83:2717-2725; Javanbakht, M. et al., 2000. J. Clin. Endocrinol.Metab. 85:2395-2401), but the skin patch causes topical skin irritationin many women, making its use problematic.

The present invention involves use of a testosterone formulated as a gelin a concentration that is appropriate for women. The data have shownthis formulation to provide effective systemic delivery of testosteronein patients with fibromyalgia. 28 days of therapy with 0.75 g1% (w/w)testosterone gel per day raised serum concentrations of total and freetestosterone in fibromyalgia patients to concentrations approximatingthose in premenopausal women. At this dose, patients showedsignificantly decreased fibromyalgia-related muscle pain, decreasedstiffness, decreased fatigue and increased libido in response totestosterone therapy. Fibromyalgia-related tender point pain wasdecreased, as well. These results, from both the pharmacokinetic andfibromyalgia-related pain assessment standpoints, support the use oftestosterone replacement therapy to treat individuals with fibromyalgiasyndrome.

Accordingly, androgen therapy provides a useful means for alleviatingsymptoms associated with FMS or CFS in women preferably of peri/postmenopausal age. By peri/postmenopausal age it is most often meant to beapproximately 40 to 60 years of age. Women outside of this range mayalso benefit since these syndromes have been known to be present inwomen 20 to 60 years of age. In a preferred embodiment, the androgenadministered comprises testosterone, an active metabolite oftestosterone such as dihydrotestosterone or androstenedione or atestosterone derivative such as methyltestosterone, testosteroneenanthate or testosterone cypionate. Examples of available pharmacologicpreparations of androgens believed to be useful in this inventioninclude, but are not limited to danazol, fluoxymesterone, oxandrolone,methyltestosterone, nandrolone decanoate, nandrolone phenpropionate,oxymethalone, stanozolol, methandrostenolone, testolactone, pregnenoloneand dehydroepiandrosterone (DHEA).

In the present invention, the androgens are administered transdermallyin a gel formulation. This formulation has advantages over current oralmethods as well as transdermal patch methods that include improvedbioavailability and a low side effect profile. In a preferredembodiment, a combination of androgens such as testosterone or atestosterone derivative and DHEA can be administered to alleviate boththe muscular and neurological symptoms of FMS or CFS.

As will be obvious to those of skill in the art upon this disclosure,other pharmaceutically acceptable androgen therapies can be considered.However, effective amounts and routes by which the androgen orcombination of androgens can be administered amount to a significantchallenge based on the number of failures in this field. Finding theright amount, formulation, and route for human use and then proving thesafety and efficacy of the formulation has been a major challenge forthose skilled in the art based on their experience with androgentherapies.

Generally, the androgen used in the invention is a biologically activeandrogen. Androgens may be active in their native state, and/or may be aprecursor or pro-drug that is metabolized to a active state upondelivery to the subject. The androgen may be, but is not limited to,testosterone, androstenedione, androstendiol, dehydroepiandrosterone,danazol, fluoxymesterone, oxandrolone, nandrolone decanoate, nandrolonephenpropionate, oxymethalone, stanozolol, methandrostenolone,testolactone, pregnenolone, dihydrotestosterone, methyltestosterone,androgen precursors, or testosterone esters.

Administration of an androgen that achieves blood levels outside thereference range can result in adverse effects. Females are especiallysusceptible because their normal blood levels are one tenth those ofmales. Levels in excess of the upper limit of the reference range +/−SEMcan cause, for example, hirsutism, acne, rapid (and sometimes permanent)changes in voice, emotional changes, and the more serious side effectsof heart disease, cancer, and liver disease. Examples of these changesand side effects are discussed below.

It is desirable to maintain a patient's androgen blood levels within themiddle to upper portion of the appropriate reference range. If androgensare administered in excess, clinical symptoms of androgen excess canappear. These symptoms can range from upsetting to dangerous. Generally,the symptoms of androgen excess include, but are not limited to,excessive and abnormal hirsutism, increased moodiness, anger, adverselipid changes, abnormal liver function, weight gain, acne, alteration oflibido, edema, functional and structural liver damage, cancer, permanentchanges in voice, emotional changes, and diabetes.

For assessing safety, female patients will be dosed adjusted such thatthe “upper limit of the reference range for free testosterone” isdefined as less than or equal to “about” 3.3 pg/mL when using the DSLfree testosterone blood level assay (Table 1); and less than or equal to“about” 19 pg/mL when using the Mayo Medical Labs free testosteroneblood level assay. If using another test with its own reference range, aperson of skill would assess safety blood level limits in a equivalentway.

High testosterone levels above the reference range have been shown toresult in the harmful effects of testosterone. Females with virilizingovarian tumors provide a good example. Regnier et al., (2002 J. Clin.Endocrinol. Metab. 87(7): 3074) disclose a case study of a woman havinga virilizing ovarian tumor (one that secretes testosterone and resultsin hyperandrogenism in about 80% of cases), with hirsutism that gotprogressively worse over time. Her total testosterone level was between3.9 ng/mL and 7.0 ng/mL, which is above the reference range for females.Once the tumor was removed, her testosterone level returned to normal,the hirsutism subsided, and the hyperandrogenism did not recur.

It has also been shown that women with certain conditions, includingbreast cancer, have a total testosterone level higher than the referencerange for women. For example, it has been shown that post-menopausalbreast cancer patients can have a total testosterone level of about 1.55ng/mL, which is above the reference range. Women with testosteronelevels of over 1.55 ng/mL have a four-fold greater chance of developingbreast cancer. (Ho et al., 2009 Singapore Med. J. 50(5):513).

Further, female-to-male (FTM) transsexuals, who are dosed withtestosterone such that their blood levels reach male levels, i.e., >300ng/dL (>3 ng/mL) have been studied for the safety of these levels inthese genotypic females. Jacobeit (2009 Eur. J. Endocrinol. 161: 795)discloses dosing females to achieve stable serum testosterone levelswithin the eugonadal male reference range of about 620+/−130 ng/dL(6.2+/−1.3 ng/mL) for 36 months. Gooren et al., (2008 J. Sexual Med. 5:765) teaches that the female-to-male transsexuals receiving doses oftestosterone at or above the male reference range develop hirsutism andmale-like increased risk for cardiovascular disease and diabetes.Bachmann et al. (2002 Fertil. Steril. 77(4): 660) teach thattestosterone and other androgens have many detrimental side effects.Specifically, testosterone can cause acne, weight gain, excess hair,increased anger, adverse lipid changes, and abnormal liver function.Franke et al. (1997 Clin. Chem. 43(7): 1262) disclose thatover-administration of anabolic steroids can cause many health problems,including weight gain, acne, hirsutism, alteration of libido, edema,function and structural liver damage. Finally, testosterone and otherandrogens are Schedule C-111 controlled substances under the AnabolicSteroid Control Act and, as such, can be dangerous to over-administer inview of the dangerous side-effects of cancer, liver disease, and cardiacdisease.

All of the above studies show the challenge to treating both women andmen with testosterone or other androgens. It is of great importance tomake sure that the dosage administered to women and men brings theandrogen level to the proper and appropriate range, or the equivalentsafe and effective range based on the detection assay used (see Table1).

By “safe,” it is meant that blood levels are not raised significantlyabove the upper end of the reference range. By “effective,” it is meantthat androgen therapy raises baseline blood levels from the lower halfof the reference range to significantly higher blood levels that arestill safe within the reference range. While total testosterone is afactor when considering the blood levels of testosterone, it is the freetestosterone that is an indicator of the testosterone that is availablefor biologic action in vivo. Further, free and bioavailable testosteronegenerally remain in a constant ratio and are reliable indicators ofbiologic availability, while SHBG-bound testosterone, which is notbioavailable, varies in response to changes in the total pool (Felig, P.and L. A. Frohman “Endocrinology and Metabolism” McGraw Hill, 4thedition, 2001 p647).

The reference ranges for women and men differ by about ten times. Table1 below, which shows the reference ranges for both women and men, andmeasured by two different detection means (male reference ranges onlyshown using one testing method). For example, the Diagnostic SystemsLaboratories (DSL) reference range for women is about 0.1 ng/mL to about1.0 ng/mL. The reference range determined using the Mayo MedicalLaboratories diagnostic test is from about 0.08 ng/mL to about 0.6mg/mL. The reference range for men, as calculated using the Mayo MedicalLaboratories diagnostic test is about 2.4 ng/mL to about 9.5 ng/mL. Itis important to remember that, when comparing serum testosteronereference ranges, one must translate the reference range from one testto another. One of skill in the art would know that diagnostic testsvary in their reference ranges, according to which, and whether,monoclonal antibody (mAb) was used for detection (earlier detectionsystems such as DSL use a detection mAb), or whether no mAb was used fordetection (more recently developed detection systems such as MayoMedical Labs, which use tandem mass spectrometry for inspectioninstead). Thus, the upper end of a safe total testosterone blood levelrange would be at about 1.0 ng/dL when using the DSL test, versus about0.6 ng/dL when using the Mayo Medical Labs test. The upper end of a safefree testosterone range would be at about 3.3 pg/mL when using the DSLtest, versus about 19 pg/mL when using the Mayo Medical Labs test.Furthermore, the reference range is only an approximation of what wouldbe the “normal” range in individuals, since the reference range would beskewed downward if the “control” population included significant datafrom subjects with a deficiency.

TABLE 1 Testosterone serum reference ranges DSL ref range¹ Mayo refrange¹ Female TT² 0.1-1.0 ng/mL 0.08-0.6 ng/mL (age 40-60 yr) (10-100ng/dL) (8-60 ng/dL) (100-1000 pg/mL) (80-600 pg/mL) Female BioT Notdetermined. 0.008-0.100 ng/mL (age 20-50 yr) (0.8-10 ng/dL) (age >50 yrunkn) (8-100 pg/mL) Female FT² 0.0003-0.0033 ng/mL 0.003-0.019 ng/mL(pre-menopausal) (0.03-0.33 ng/dL) (0.3-1.9 ng/dL) (0.3-3.3 pg/mL) (3-19pg/mL) % of TT that is BioT 10-17% of TT is BioT % of TT that is FT 3-4%of TT is FT % of BioT that is FT 19-38% of BioT is FT Male TT² 2.4-9.5ng/mL (age >18 yr) (240-950 ng/dL) (2,400-9,500 pg/mL) Male BioT0.61-2.13 ng/mL (age 40-49) (61-213 ng/dL) (610-2,130 pg/mL) Male2.0-4.8% of TT is FT % of TT that is FT [~22% of TT is BioT] Male FT0.09-0.3 ng/mL (9-30 ng/dL) (90-300 pg/mL) Abbreviations: TT = totaltestosterone (free testosterone + testosterone weakly bound to albumin +testosterone tightly bound to sex hormone binding globulin SHBG); FT =free testosterone (unbound) BioT = bioavailable (or bioactive)testosterone (free testosterone + testosterone weakly bound to albumin)¹Because reference ranges vary according to the antibody used in thetest, the source of reference ranges used for the values in this tableis indicated: DSL (Diagnostic Systems Laboratories); Mayo (Mayo MedicalLaboratories), a common testing service in hospitals for testing TT, FTand BioT (analysis by tandem mass spec after AmSO4 precipitation).Claims based on the reference range from an antibody detection test suchas DSL must be converted to the Mayo Medical Labs reference range, whichdoes not rely on antibody detection of testosterone, in order to makecomparisons. The above table can be used for this purpose. ²Maletestosterone levels are generally on the order of 10x femaletestosterone levels; Free testosterone is on the order of 1-5% of totaltestosterone.

In one embodiment of the invention, and for both the method of treatingfibromyalgia-related pain in a female human, the androgen can betransdermally administered in a daily unit dose of about 0.1 mg to about12.8 mg of the androgen in a pharmaceutically acceptable carrierformulated for daily topical administration as a gel and wherein the gelis formulated to deliver steady state total androgen serum levelswithout raising free androgen serum levels or twenty-four hour freeandrogen AUC above the levels required for therapeutic efficacy andsafety. Preferably, the daily unit dose of the androgen is from about1.0 mg to about 12.8 mg. More preferably, the daily unit dose of theandrogen is from about 2.5.0 mg to about 10.0 mg. More preferably, thedaily unit dose of the androgen is from about 3.2 mg to about 9.6 mg.Even more preferably, the daily unit dose can be from about 4.4 mg toabout 9.6 mg. Most preferably from about wherein the daily unit dose ofthe androgen is from about 6.0 mg to about 8.0 mg. The daily unit doseof the androgen can be about 6.5 mg or about 7.5 mg.

In another embodiment, the dosing range can be incremental. For example,the dose to be administered to a female subject can be about 2.5 mg; 5.0mg; 7.5 mg; or 10.0 mg. In order to determine the most appropriatedosage for a particular subject, a physician may start the patient on alow dose, and titrate the dose upwards until an dose that is botheffective and safe is reached. In yet another embodiment, theincremental dosage rate can start at 3.2 mg, and rise progressively to6.4 mg, 9.6 mg, and 12.8 mg, using a 0.8% gel formulation. For example,patients can be started with 2 packets of 0.8% testosterone or placebogel per day for the first four weeks. Each packet can contain 400 mg of0.8% testosterone gel (3.2 mg testosterone, to deliver 10% or 320 μgbioavailable testosterone) or 400 mg Placebo gel in it. After fourweeks, any patient who tests >3.3 pg/mL for serum free testosterone(testosterone blood levels above the reference range), can decrease thedose by one gel packet/day. Any patient who tests ≦1.9 pg/mL for serumfree testosterone (at the low end of the testosterone reference range)can increase dose by one gel packet/day until the blood level is raisedto near the mid-range or near the upper end of the reference range.

The daily unit dose can be delivered via a transdermal gel having about0.1% to about 10.0% of the androgen. Preferably, the transdermal gel canhave about 0.5% to about 5.0% of the androgen. More preferably, thetransdermal gel can have about 0.5% to about 2.5% of the androgen. Mostpreferably, the transdermal gel can have about 0.8-1.0% of the androgen.

In another embodiment of the invention, and for treatingfibromyalgia-related pain in a female human subject, the daily unit doseof the androgen may be selected to maintain steady state total androgenserum levels within a range of between about 0.7 ng/mL and about 1.6ng/mL, and preferably between about 0.9 ng/mL and about 1.4 ng/mL for atleast 24 hours after administration without raising free androgen serumlevels or twenty-four hour free androgen AUC above the levels requiredfor therapeutic efficacy and safety. Further, the free androgen serumlevels and twenty-four hour free androgen AUC should not be raised abovelevels required for therapeutic efficacy and safety.

Specifically, the free androgen serum levels can be raised to betweenabout 1.0 pg/mL and about 3.5 pg/mL. (About 3.3 pg/mL using the DSL testis equivalent to about 19 pg/mL using the Mayo Medical Labs massspectrophotmetry method of measuring free testosterone, Table 1. Theupper end of the reference range is determined by which method ofmeasuring free testosterone is used). The twenty-four hour free androgenAUC levels can be raised to between about 35.18 pg-h/mL and about 72.60pg-h/mL; more preferably the free androgen serum levels can be raised tobetween about 2.00 pg/mL and about 3.3 pg/mL and the twenty-four hourfree androgen AUC levels can be raised to between about 40 pg-h/mL andabout 65 pg-h/mL The daily unit dose of the androgen can be from about4.0 mg to about 10.0 mg, or from about 6.0 mg to about 8.0 mg. At 10%bioavailability for the androgen in the gel that is actually deliveredto the blood, the daily unit dose-to-be-delivered of the androgen can befrom about 0.4 mg to about 1.0 mg, or from about 0.6 mg to about 0.8 mgandrogen.

The formulation can be prepared using a variety of pharmaceuticallyacceptable ingredients. For transdermal administration, the formulationcan include, but is not limited to, the androgen, a penetrationenhancer, an emulsifier, a gelling agent; a lubricant, a thickeningagent, a buffer, an alcohol, and water. As will be evident to those ofskill in the art upon this disclosure, other pharmaceutically acceptableandrogen therapies can be used. Effective amounts and routes by whichthe androgen or combination of androgens can be administered in a safeand effective manner according to the present invention can also beused, such that safe and effective blood levels of androgen areobtained. The formulation will preferably be used at a unit dose of 800mg gel of 0.8% testosterone (6.4 mg testosterone), and then, dependingon blood levels at 4 weeks, adjusting down to 400 mg gel of 0.8%testosterone (3.2 mg testosterone), or adjusting up to 1200 mg gel of0.8% testosterone (9.6 mg testosterone), to maintain the unit dose toachieve safe and effective blood levels. Administration of thecomposition of the invention results in a low side-effect profile anddelivers a therapeutically effective daily amount of the androgen to thepatient's serum over each 24 hour period to alleviate the patient'ssymptoms without causing androgenic side effects.

The composition of the present invention comprises, in addition to theaforementioned androgen/anabolic agent, co-treatment with apharmaceutically effective amount of growth hormone elicitor oreffector, either growth hormone or an agent that is known to releasegrowth hormone in effective amounts, i.e., a growth hormone releasingagent (“GRF”). GRF is an acronym based on the existence of an endogenoushormone known as GHRH. Other agents include GHrelin or a growth hormonereleasing peptide or analog (GHRP; GHRP-6, or hexarelin,His-DTrp-Ala-Trp-DPhe-Lys, and GHRP-2, or Dala-D-2-NaI-Ala-Trp-Dphe-Lysare examples), which have been shown to release effective amounts ofgrowth hormone. The natural rhythm of growth hormone release from thepituitary gland results in release of insulin-like growth factor(IGF-1), which in general, is considered to be the causal agent thatdetermines the course of hormonal regulation and balance in processessuch as adipogenesis and myogenesis. The hormonal effector, then, forthe purpose of this invention, is also prophetically considered to beany peptide or peptidomimetic agent that directly acts to release thissecondary anabolic growth factor, (IGF-1), not necessarily through theintermediary route of secretion of growth hormone itself. Although theindirect growth hormone route is preferred to elicit IGF-1, the latterroute to directly release IGF-1 also is included by example.

In another embodiment of the present invention, the compositioncomprises a pharmaceutically effective amount of a growth hormone or,more preferably, a growth hormone-releasing agent, or an elicitor ofIGF-1 secretion, coupled with androgen treatment and such combinedtreatment being capable of counteracting the deleterious effects ofaging, such as, for example, muscle weakness, body fat increases, andskin fragility in adults. Essentially any suitable growthhormone-releasing agent may be employed in combination with anyandrogen, preferably one such as testosterone that possesses stronganabolic activity. Other anabolic agents that are not thought of asandrogenic agents, or do not possess maximal androgenic activity may beused, as long as they have appreciable anabolic activity. In fact, thisinvention anticipates, and includes as a prophetic example, thoseanabolic agents that may be completely devoid of androgenic activity.Examples of such growth hormone-releasing agents include:somatoliberins; growth hormone-releasing hormone active fragments, suchas, for example, hGRF (1-29) amide and hexarelin (GHRP-6). Hexarelin isa growth hormone releasing peptide mimetic agent, i.e., it mimics theeffects of growth hormone releasing peptide in the body and containsbetween 2 and 20 amino acids. In particularly preferred embodiments,more than one growth hormone-releasing agent may be used in combination.A preferred combination comprises growth hormone-releasing factor (GRFor GHRH) and a growth hormone releasing peptide or peptidomimetic(GHRP). This combination has been reported to act by separate mechanismsfor the release of endogenous growth hormone, and the effects have beenshown in some cases to be additive, or even, synergistic, working at aseparate receptor often called the Ghrelin receptor, to differentiate itfrom the GHRH receptor. Since the GHrelin receptor has recently beenelucidated, prophetically other ligands for this receptor areanticipated to be synthesized and/or discovered in the future, and theseare included by example (Baldelli, R el. al., Endocrine 14 (1):95-99,2001). These are often referred to as GHSs (growth hormonesecretagogue).

The administration of a GH or IGF-1 secretagogue will reduce plasmaandrogen concentration in humans (Tapanainem J. et al., Fertility andSterility 58: 726-732). This effect increases the need for exogenousandrogen, such as testosterone, to be also administered as aco-treatment to restore and amplify existing levels.

Other compounds are known to affect this system which is known as thehypothalamo-pituitary-hepatic axis for GH, among other terms.Prophetically, it is probable that other compounds involved in thishormonal regulatory system may play a role in indirectly or directlyinfluencing and increasing levels of GH, IGF-1, or IGF-2, and may beadministered in the context of this invention along with the androgenicsupplementation to get maximal effects of the growth/anti-aging effectsof such treatment. Other indications that may be treated besidesfibromyalgia may be syndromes affecting the growth of individuals,including but not limited to pituitary dwarfism, conditions or syndromesthat are well known to practitioners in the field of endocrinology,growth, and aging.

For the administration of the GH agents that are described in detailabove, they may be administered by a variety of means. These agents maybe administered separately from the androgen administration, using themodalities of intranasal, transdermal, parenteral (subcutaneous orintravenous), or oral (with or without permeation enhancement andpreferably with enteric protection, since proteins and peptides may bedegraded by gastric exposure). GH itself is most preferably administeredby parenteral means in practice, because it is a large protein that isof limited stability and limited absorption. However, intranasaladministration is also an acceptable means for this and other largeproteins or peptides.

In addition to a separate delivery modality for the GH agent and theandrogenic compound selected for treatment, the two may be combined in asingle combination therapy. For example, both could be incorporatedtogether in an oral form, tablet, or suspension, with the caveat thatany proteinaceous agent is suitably protected from gastric degradation.Alternatively, the combination of agents may be administeredintranasally in one unit through separate delivery chambers, known tothose of skill in intranasal delivery, or together in the same liquid,semi-solid, or solid delivery form. For example, a microparticulate ornanoparticulate dry solid system could be administered intranasally. Orthe combined agents could be both administered transdermally.

Example 1

A clinical trial was performed to investigate the pharmacokinetics andefficacy of transdermal delivery of hormones for treatment offibromyalgia, the data from which can be used to plan further studiesrelating to fibromyalgia-related pain. Women were recruited byinstitutional review board-approved advertising. Subjects aged 40-55 anddiagnosed for fibromyalgia using American College of Rheumatologycriteria (11/18 bilateral tender points above and below the waist,chronic fatigue, etc., (Wolfe, F. et al., 1990. Arthrit. Rheumat.33:160-172) were selected for the study if they fit additional criteria.Women were included if, in addition to meeting all other criteria, theyagreed to keep their medicines unchanged during the study (decreases inanalgesics were permitted). Women taking hormone replacement therapywere enrolled if they agreed to come off hormone therapy at least 2weeks prior to, and for the duration of, the study, in addition tomeeting other eligibility criteria. Pre- or peri-menopausal women wererequired to have adequate alternative contraception, a negativepregnancy test, and treatment was started within the follicular(proliferative) phase of the menstrual cycle. Patients were included ifthey were willing to exercise 20 minutes a day, 5 days per week duringtherapy, to promote the effects of testosterone; this was a requirementput in place by the Institutional Review Board. It is noted thatexercise can be difficult for fibromyalgia patients. It is predictedthat exercise alone cannot provide the therapeutic benefit oftestosterone replacement therapy; a placebo-controlled study willconfirm this prediction.

In this study testosterone was formulated as a gel in a concentrationthat is appropriate for women, and showed effective systemic delivery oftestosterone in patients with fibromyalgia. The patients received 28days of therapy with 0.75 g1% (w/w) testosterone gel once per day. Serumconcentrations of total and free testosterone were raised in thesefibromyalgia patients to concentrations approximating those inpremenopausal women. At this dose, patients showed significantlydecreased fibromyalgia-related muscle pain, decreased stiffness,decreased fatigue and increased libido in response to testosteronetherapy. Fibromyalgia-related tender point pain was significantlydecreased, as well. These very encouraging results, from both thepharmacokinetic and fibromyalgia-related pain assessment standpoints,support the use of testosterone replacement therapy to treat individualswith fibromyalgia syndrome in a formal double blind placebo-controlledstudy. Further, the conclusions of efficacy in patients withfibromyalgia syndrome in response to increased serum concentrations oftestosterone parallel the positive outcomes found with testosteronereplacement therapy in other populations with chronic fatigue and musclewasting, e.g., AIDS and Parkinson's Disease.

Children, pregnant women, and women on hormone therapy, hormonecontraceptives or infertility drugs were excluded. Women were excludedfrom the study if they reported undiagnosed vaginal bleeding, had a bodymass index BMI>30, admitted to ethanol or illicit drug abuse, had activethrombophlebitis, breast cancer, hypertension (BP>160 systolic/95diastolic with or without medication, after sitting 5 minutes), or majorskin disease, acne or hirsutism.

Prior to enrollment, study patient blood was tested for the followinggeneral health criteria (exclusion criteria in parentheses): cardiacrisk factors by lipid profile—total fasting cholesterol (>240 mg/dL),high density lipoprotein (<35 mg/dL), low density lipoprotein (>210mg/dL), triglyceride (>300 mg/L); hepatic function by alanineaminotransferase (>1.5×N; normal at 0-40 U/L), alkaline phosphatase(>2×N; normal at 40-120 U/L), aspartate aminotransferase (>1.5×N; normalat 10-30 U/L), serum albumin (>N; normal at 3.2-5.2 g/dL), totalbilirubin (>N; normal at 0.2-1.3 mg/dL), and direct (conjugated,soluble) bilirubin (>N; normal at 0.0-0.3 mg/dL); kidney function byblood urea nitrogen (>2×N; normal at 8-18 mg/dL) and serum creatinine(>N; normal at 0.7-1.2 mg/dL) tests; hematological function was assessedby complete blood cell count including testing for hemoglobin (normal;12-16 g/dL).

Blood tests and physical exam at the end of the study were performed toassess whether testosterone therapy adversely affected the generalhealth of the study patient. Serum total testosterone (>0.4 ng/mL) andFSH (<22 IU/L) were tested as well (8:00 AM after overnight fasting), toconfirm patients had concentrations of testosterone in the lower half ofthe reference range (2 patients out of 18 were excluded based ontestosterone concentrations) and to determine their post-menopausalstatus. FSH concentrations <22 IU/L indicated premenopausal orperi-menopausal status and thus the need for adequate contraception,unless the patient had undergone bilateral oophorectomy. Testosteroneserum concentrations were tested at 8:00 AM due to the small circadianrhythm of circulating androgens. The most frequent exclusion criterionwas for BMI >30. Patients were required to stop taking St. John's wort,since St. John's wort is known to induce catabolism of hormones byactivating CYP3A, a detoxifying enzyme complex in the liver.

Written informed consent was obtained from study subjects prior to entryinto the study. This consent process was ongoing throughout the study,and an independent Data Safety Monitoring board was in place for theduration of the study. Twelve patients who fit the eligibility criteria,above, were scheduled for physical exams including tender pointassessment, verification of fibromyalgia diagnosis, and assessment ofgeneral health.

On day 1, blood was drawn by venipuncture at 0, 1, 2, 3, 4, 6, 8, 10, 12and 24 hrs for 24 hr pharmacokinetic profiling of baseline testosteroneserum concentrations. Testosterone gel, 0.75 g1% w/w, was applied by thepatients to their lower abdominal skin just after the zero time pointblood draw (8:00 AM). The patients also filled out afibromyalgia-related pain assessment questionnaire form and were givenpackets of testosterone gel for 8:00 AM daily application to lowerabdominal skin, instructions for use and a patient medication log andexercise log for 28 days of therapy. On day 28, the blood draws for 24hr pharmacokinetic profiling were repeated, and a follow-up exam wasrepeated at the end of the 28 days of therapy.

The delivery vehicle for this study was a gel formulation. A goal of thestudy was to identify a transdermal delivery system for hormones thatwould result in effective levels of hormones in blood as a way to reduceside effects of androgen therapy. The gel used for this study was a 1%w/w testosterone gel, USP grade. The once-a-day daily unit dose appliedwas about 7.5 mg testosterone; the expected bioavailability of 10%,industry standard for transdermal delivery, would deliver about 0.75 mgtestosterone over 24 hr. The gel was formulated for women by BentleyPharmaceuticals, Inc. (Exeter, N. H., a former division of CPEXPharmaceuticals, Inc.) using good manufacturing practice standards, andis quick-drying, odorless, colorless, comfortable on the skin, andnon-staining. Since testosterone is a Schedule C-III controlledsubstance under the Anabolic Steroid Control Act, all testosteronetreatment samples were itemized and accounted for at the conclusion ofthe study.

Testosterone concentrations were determined by enzyme linked immunoassay(EIA, Diagnostic Systems Laboratories or DSL, Inc, Webster, Tex.), whereserum testosterone from study subjects competed with enzyme-linkedtestosterone bound to anti-testosterone mAb. This assay system wasdesigned to detect the lower concentrations of testosterone found inwomen as well as concentrations in the upper ranges. Free testosteroneconcentrations were determined by EIA using an anti-testosteroneantibody that recognizes the unbound testosterone in the test sample,and has low affinity for sex hormone binding globulin and albumin.Reference ranges for the DSL detection system are given in Table 1above. For the purposes of determining mean testosterone concentrations,times were based on the nearest hour. Of the 240 time points taken forthe pharmacokinetic data (10 time points per individual x 2 sets perindividual x 12 individuals), 1 time point was missed (4 hr point) and 3additional time points were in between the standard times for takingblood (8 hr point; 4 hr and 10 hr points). Values for these time pointswere derived by interpolation for the purposes of deriving meantestosterone concentrations. A noncompartmental pharmacokinetic analysisusing WinNonlin Pro (Pharsight, Mountain View, Calif.) used the exacttime points recorded for all the patients.

In order to determine the efficacy of the treatment for reducingsymptoms of fibromyalgia, patients filled out questionnaire forms on day1 and again at the end of therapy on day 28 to assessfibromyalgia-related pain. The patient questionnaire was based on apublished and validated Fibromyalgia Impact Questionnaire as well asother accepted criteria for fibromyalgia patient assessment (Wolfe, F.1990. Arthrit. Rheumat. 33:160-172; Goldenberg, D. 1996. Arthrit.Rheumat. 39:1852-1859; Burckhardt, C. S. 1991. J. Rheumatol.18:728-733), and used a 100 mm visual analog scale (VAS). Tender pointexams were administered by a qualified rheumatologist experienced intreating women with fibromyalgia, and involved applying approximately 9pounds of pressure at each tender point and asking whether the patientfelt fibromyalgia-related pain. This practice is in accordance withcriteria specified by the American College of Rheumatology. Exams wereadministered just prior to Day 1 of therapy (and therefore designated as“pretreatment”), and at the end of therapy. The pretreatment tenderpoint assessment was performed on all patients within 1 week before thestart of therapy. Dolorimeter readings were taken from the bilateralsecond costochondral junction and trapezius tender points, forcomparison, in 11 of the 12 study subjects.

Pharmacokinetic analysis of serum testosterone concentration data wascarried out using WinNonlin Pro software, using the noncompartmentalmodel with extravascular input. Differences between Day 1 and Day 28maximum plasma concentrations (C_(max)) and area under the curve (AUC)of a plot of plasma concentrations over time were assessed bycalculating individual subject Day 28 minus Day 1 data and estimating95% confidence intervals of this difference to determine if significance(p<0.05) was reached. Tender point data evaluations were analyzed byStudent's t test (paired, 2-tailed) after summing all 18 tender pointvalues for each individual at baseline versus day 28 of the study.

Eighteen patients were screened and evaluated for enrollment in thestudy. Twelve fibromyalgia patients aged 40-55 who met the studyeligibility criteria were enrolled for this study and treated. Allpatients were white Caucasian females. Patient demographics for age,height and weight are shown in Table 2 below.

TABLE 2 Patient Demographics Pat ID# Age Ht (in) Wt (lb) BMI 001 54 66155 25.1 002 54 62 139 25.9 005 51 64 150 26.0 006 53 60 130 25.5 007 5462 160 29.3 009 53 67 160 25.1 010 45 67 175 27.5 011 50 62 135 24.8 01254 62 122 22.4 016 55 64 140 24.3 017 45 62 140 25.7 018 42 64 130 22.4Mean 51 63 145 25.3 Median 53 63 140 25.3 Minimum 42 60 122 22.4 Maximum55 67 175 29.3

Analysis of the blood testosterone concentration data revealed thatserum total testosterone concentrations were reliably increased infibromyalgia patients in response to testosterone gel hormonereplacement therapy. Serum total testosterone concentrations versus timedata for Day 1 and Day 28 are shown in FIGS. 1A-1C. Reference ranges forthe concentration of total testosterone in serum from women aged 40-50and 50-60 are provided in Table I above and denoted by dashed horizontallines in FIGS. 1A-1C.

FIGS. 1A-1C relate to the serum total testosterone concentrations infibromyalgia patients and show that these concentrations are increasedin response to testosterone gel therapy. Blood was taken by venipuncturefrom twelve fibromyalgia patients who fit the eligibility requirements,on day 1 at the times indicated and then again on day 28 of therapy. Lowand high ends of the reference range are as indicated by the dashedhorizontal lines, and as specified in Table 3. FIG. 1A shows the day 1profile, FIG. 1B shows the day 28 profile, and FIG. 1C shows the means±SEM for day 1 (open symbols) versus day 28 (filled symbols).

The day 1 zero time point for total testosterone confirmed that thesepatients initially had total testosterone concentrations in the lowerhalf of the reference range. The mean serum concentration of totaltestosterone 24 hr after application of the first dose of hormone on Day1 was significantly higher than the mean serum concentration for timezero on Day 1 (FIG. 1C, p=0.01), indicating that serum concentrationsreached steady state very quickly, within 24 hr, in the populationaggregate during the 28 day time course. Testosterone concentrationversus time data, therefore, did not permit estimation of half-life.Steady state concentrations were maintained by day 28, as evidenced bythe similar mean concentrations at the beginning and end of the 24 hrsampling. Interestingly, and not unexpectedly, substantial variation wasfound in the 24 hr profiles on an inter-individual basis, consistentwith the complex regulation known for this hormone.

Summary pharmacokinetic parameter analysis demonstrated significantlyincreased mean total testosterone maximum concentration in response totestosterone therapy: C_(max) was 1.92 ng/mL +0.90 SD on day 28 comparedwith 1.21 ng/mL ±0.71 SD on day 1, p<0.05 (Table 3 below). Significantlyincreased mean total testosterone area under the curve values (assessedover the 24 hr profiling time period) were also found: AUC was 28.75ng-h/mL +13.91 SD on day 28 compared with 18.36 ng-h/mL 7.10 SD on day1, p<0.05. The differential C_(max) and AUC values for day 28 aftersubtraction of day 1 baselines are provided in the right panel of Table3.

TABLE 3 Total Testosterone Pharmacokinetic Parameters Total Testosteroned1 (n = 12) Total Testosterone d28 (n = 12) d28-d1 Differential T_(max)C_(max) C_(min) AUC_((0-24 hr)) T_(max) C_(max) C_(min) AUC_((0-24 hr))C_(max) C_(min) AUC_((0-24 hr)) h ng/mL ng/mL ng · h/mL h ng/mL ng/mL ng· h/mL ng/mL ng/mL ng · h/ml Mean 1.21 0.17 18.36 1.92 0.72 28.75 0.70.55 10.38 SD 0.71 0.10 7.10 0.90 0.44 13.91 0.96 0.42 12.61 Median16.11 1.08 0.18 16.88 10.17 1.79 0.61 26.71 Min 2.33 0.53 0.00 10.351.62 0.63 0.21 11.29 Max 24.75 3.05 0.37 30.95 23.92 3.13 1.67 59.56 95%CI high -> 1.36 0.79 19.23 CIs CI low -> 0.06 0.31 1.55 For Diff

The pharmacokinetic data for total testosterone, all together,demonstrate that with therapy, mean serum total testosteroneconcentrations initially rose quickly over the first 3 hours and werethen reliably sustained over time. In addition, mean serumconcentrations were raised from the lower boundary of the referencerange to about the upper end of the reference range for premenopausalwomen.

Low free testosterone concentrations in serum are raised topremenopausal concentrations in fibromyalgia patients in response totestosterone gel hormone replacement therapy.

TABLE 4 Free Testosterone Pharmacokinetic Parameters Free Testosteroned1 (n = 10) Free Testosterone d28 (n = 10) d28-d1 Differential T_(max)C_(max) C_(min) AUC_((0-24 hr)) T_(max) C_(max) C_(min) AUC_((0-24 hr))C_(max) Cmin AUC_((0-24 hr)) h pg/ml pg/mL pg · h/ml h pg/ml pg/mL pg ·h/ml pg/ml pg/mL pg · h./ml Mean 2.64 0.54 35.0 3.91 1.29 53.89 1.270.75 18.9 SD 1.70 0.41 18.3 1.23 0.54 18.71 1.71 0.72 27.1 Median 6.382.19 0.41 31.8 9.13 4.16 1.12 51.49 Min 1.92 0.95 0.09 13.7 0.00 1.810.71 27.95 Max 24.8 6.86 1.35 64.1 24.05 5.58 2.27 90.08 95% CI high ->2.33 1.19 35.6 CIs CI low -> 0.21 0.30 2.10 For Diff

Concentrations of free testosterone in serum were analyzed similarly tototal testosterone, with the results shown in FIGS. 2A-C. Serum freetestosterone concentrations were quantitated using methodology similarto FIGS. 1A-1C. The low end of the reference range for this study forpre- and post-menopausal women was 0.33 pg/mL, represented by the lowerdashed horizontal line. The high end of the reference range forpremenopausal women is represented by the upper dashed horizontal line,and the high end for post-menopausal women is represented by the middledashed horizontal line (FIG. 2 and Table 1). FIG. 2A shows the day 1profile. FIG. 2B shows the day 28 profile. FIG. 2C shows the means±SEMfor day 1 (open symbols) versus day 28 (filled symbols), with a changein the y-axis scale. Patient IDs are provided in the legend for FIGS. 2Aand 2B.

These results were similar to the previous findings, but with thefollowing particular findings. Two individuals had unusually highconcentrations of free testosterone prior to, and throughout, the courseof therapy despite normal total testosterone levels, suggesting seruminterference with the free testosterone testing antibody in these twocases. (The only medication and/or supplement reported by both studysubjects, and not by any other subjects, was ginger root, and theanti-depressant Trazodone was taken by both individuals. Preliminarydata, from spiking the assay with ginger root or Trazodone, areconsistent with interference by ginger root and/or Trazodone with theenzyme linked immunoassay for free testosterone: 1) for aproof-of-concept finding, others have shown that DHEA-S can interferewith testosterone immunoassays; 2) here, day 1, 0 hr baseline freetestosterone blood levels were higher than was biologically reasonableand did not increase further with therapy; 3) these two individuals hadlow total testosterone baseline levels such that if the baseline freetestosterone levels were real, a much larger fraction of the totaltestosterone would have been free testosterone than the normal 1-4%; 4)the free testosterone for these individuals was about 3-10× thereference range, a level that approaches the levels found in males; 5)these individuals would have been at high risk for hirsutism if thesefree testosterone blood levels were indeed so much higher than thereference range; and 6) this study excluded patients with hirsutism. Forthese reasons, these two outliers were deleted from the analysis).Individual profiles for the remainder of the patients showedconcentrations that increased from the post-menopausal range to thepremenopausal and upper post-menopausal reference range.

Summary pharmacokinetic parameter analysis showed a mean freetestosterone C_(max), of 4.69 pg/mL +2.17 SD on day 28 compared with3.68 pg/mL ±2.99 SD on day 1 (p>0.05) [adjust for outliers] and a meanfree testosterone AUC of 71.38 pg-h/mL ±45.76 SD on day 28 compared with54.35 pg-h/mL ±49.83 SD on day 1 (p>0.05) (Table 4). Free testosteroneC_(max), and AUC were increased with therapy, as evidenced bysubtraction of the day 1 baseline from day 28 values, but statisticalsignificance was not achieved [adjust for outliers] in thesepharmacokinetic parameters due to the two individuals with exceptionallyhigh free testosterone concentrations.

Testosterone gel therapy is associated with a statistically significantreduction in fibromyalgia-related tender point pain comparing scores atthe beginning of therapy and at the end of therapy. These evaluationswere conducted a rheumatologist, the results are shown in FIG. 3. Tenderpoints defined in Table 4. Analysis of the tender point data showed thattransdermal testosterone gel therapy was associated with decreasedsubjective assessments of tender-point pain. Using a pain scale of 0 to10, where zero is no pain, there were mean decreases in pain for everytender point, with statistical significance achieved after summingvalues across all 18 tender points (p=0.012), a finding that comparesfavorably to studies using calcium channel blockers or SNRI therapeuticsto treat fibromyalgia patients. The results are shown in FIG. 3, whichdemonstrates that fibromyalgia-related tender point pain was decreasedin fibromyalgia patients after testosterone gel therapy. Tender pointexams were administered by the study rheumatologist.Fibromyalgia-related pain was assessed on a visual analog scale (VAS) of0-10, with zero being no pain, and 10 being the most pain. The p value,determined by summing all tender point values for each patient at thebaseline versus d28 time points and assessing by paired Student'sT-test, was p=0.012. The tender points indicated along the abscissa aredefined in Table 5. The dark bars of FIG. 3 indicate pretreatment (PT)measurements and the hatched bars indicate measurements taken on day 28.

Using a dolorimeter to assess fibromyalgia-related pain at the sameoffice visit, pain responses were quantitated for the bilateral secondcostochondral junction and bilateral trapezius tender points (n=10).FIG. 4 shows that fibromyalgia-related tender point pain threshold wasincreased in fibromyalgia patients after testosterone gel therapy.Tender points were assessed independently by dolorimeter, with higherreadings indicating a higher threshold for fibromyalgia-related pain.Thus, increased values were expected in response to testosterone geltherapy. A limited number of tender points were evaluated by this methodin 10 study subjects. Individual response values ranged from 2 to 9.Mean dolorimeter values for the pressure at which patients reportedfibromyalgia-related pain were higher at the end of 28 days oftestosterone treatment, which would be expected if therapy increasedthresholds of fibromyalgia-related pain, although the dolorimetryresults did not reach statistical significance. Therapy over a longerperiod of time, with a greater number of subjects and/or over moretender points is likely to provide significance by dolorimeterassessment.

TABLE 5 TP # Tender Point Description Lay description 1-2 LowerBilateral lower cervical At the base of the neck in the back Cervical(paraspinals) at the anterior aspect of the intertransverse spaces atC5-7 3-4 Second rib Bilateral at the second On the breast bonecostochondral junction (rib- cartilage) just lateral to the junction ofthe upper surface 5-6 Lateral Bilateral lateral epicondyle On the outeredge of the forearm about epicondyle in forearm, 2 cm distal to an inchbelow the elbow the epicondyles 7-8 Gluteal Bilateral gluteal in the Onthe outside of the hip upper outer quadrant of buttock in the anteriorfold of muscle  9-10 Occiput Bilateral occiput at the At the base of theskull beside the spinal insertion of the suboccipital column muscle11-12 Trapezius Bilateral trapezius at On the top of the shoulder towardthe midpoint of the upper back (flat triangular muscle post, neck,border shoulder) 13-14 Supraspinatus Bilateral supraspinatus at its Overthe shoulder blade origin above the scapular spine near the medialborder 15-16 Greater Bilateral greater trochanter At the top of the hiptrochanter posterior to the trochanteric prominence 17-18 Knee Bilateralknee at the medial On the fat pad over the knee fat pad just proximal tothe joint line 1-8 anterior 9-18 posterior

A p value of p=0.17 for the dolorimetry data was derived using summedtender point values, similar to FIG. 3. The dark bars indicatepretreatment (PT) measurements and the diagonal bars indicate Day 28measurements.

Fibromyalgia-related pain parameters were also evaluated by patientquestionnaire using a visual analog scale (VAS) from 0-10. FIGS. 5A-5Bshow that fibromyalgia symptoms were improved in fibromyalgia patientsafter testosterone gel therapy. Patients were administered a PatientQuestionnaire Form by the study coordinator to assess their feelings ofpain. For libido, 10 represents the strongest feelings of libido (sexdrive or drive/joy in life; increases expected in response to therapy).For the remaining categories, 10 represents the worst severity ofsymptoms (decreases expected in response to therapy), with the exceptionthat headache frequency was measured on a scale of 1-4. The dark barsindicate day 1 measurements and the hatched bard indicate day 28measurements. FIG. 5A shows the parameters that are normally highlyprevalent in fibromyalgia patients and FIG. 5B shows parameters that aremore weakly prevalent in fibromyalgia patients with the exception ofsleeplessness.

Libido (sex drive) was increased in response to testosterone treatment.Muscle pain, stiffness, fatigue upon awakening and tiredness were alldecreased during testosterone treatment. For muscle pain, a definingsymptom for fibromyalgia, 42% of patients had a greater than 50%improvement in fibromyalgia-related pain. Muscle pain VAS improvementwas not dependent on whether the patient had previously had a clinicalrelationship with the study rheumatologist, and might therefore wish toplease the doctor (data not shown).

These findings are consistent with the idea that restoration ofpremenopausal serum testosterone concentrations relieves symptoms thatmost specifically relate to testosterone deficiency, e.g., loss ofsexual desire, loss of muscle function and increased fatigue. Indeed,patients reported decreased fibromyalgia-related muscle pain, decreasedstiffness, decreased fatigue upon awakening, decreased tiredness, andincreased libido. Blood tests and physical exam at the end of the study,including cardiac function, liver function and kidney function panelassessment, verified testosterone therapy did not adversely affect thegeneral health of the study patient, and no study patient reported anyadverse events that were attributable to the treatment.

Most trials involving hormone replacement therapy have used derivativesof hormones naturally found in women. These derivatized hormones havebeen promoted because of their patentability and their extended halflife. Androgens are no exception since the androgen hormone mostprescribed for women is methyltestosterone, where methylation at theC-17 position increases its oral bioavailability. Patients do nottolerate derivatized hormones very well, meaning that those hormones arenot good candidates for commercialization. Non-derivatized exogenoushormones that are structurally identical to endogenous hormones haveshort plasma/serum half lives that range from 10-100 minutes, makingoral administration of native hormones problematic. Investigators havebegun to develop transdermal delivery systems, which provide sustaineddelivery while minimizing hepatotoxicity. A testosterone skin patch hasbeen effective in HIV seropositive women with wasting syndrome (Miller,K. 1998. J. Clin. Endocrinol. Metab. 83:2717-2725; Javanbakht, M. 2000.J. Clin. Endocrinol. Metab. 85:2395-2401), but the skin patch causestopical skin irritation in about 30% of women, making its useproblematic.

This example involves use of a testosterone formulated as a gel in aconcentration that is appropriate for women. The data have shown thisformulation to provide effective systemic delivery of testosterone inpatients with fibromyalgia. 28 days of therapy with 0.75 g1% (w/w)testosterone gel per day raised serum concentrations of total and freetestosterone in fibromyalgia patients to concentrations approximatingthose in premenopausal women. At this dose, patients showedsignificantly decreased fibromyalgia-related muscle pain, decreasedstiffness, decreased fatigue and increased libido in response totestosterone therapy. Fibromyalgia-related tender point pain wasdecreased, as well. These results, from both the pharmacokinetic andpain assessment standpoints, support the use of testosterone replacementtherapy to treat individuals with fibromyalgia syndrome.

The following tables contain raw data from the study set forth above.For all these tables, the table patient numbering correlates with thePatient Study Identification Number as listed below. Patient Nos. 7 and12 (010 and 016, respectively) are outliers with regard to their freetestosterone level, and are taken into account as described in paragraph[0079].

Testosterone Bl Level PK Data Patient ID Number Study Patient ID Number1 009 2 005 3 007 4 001 6 002 7 010 8 011 9 012 10 017 11 006 12 016 13018

GENERAL HEALTH - over/counter meds d 0 ID# List 1 #pills 1 List 2 #pills2 List 3 #pills 3 001 Ibuprofen 4.5/d 002 Motrin IB 0 to 2/d 005 Advil6/wk Acetaminophen 4/wk ASA 325 mg qd1 1/d 006 Advil 2 to 4/wk Aleve1-2/wk 007 Motrin IB 2/wk 009 Advil 3/d Zantec no entry 010 Cortizone crPRN, Olocon cr 0.1% PRN, ginger rt PRN stomach aches, Vioxx 50/d;Stopped for study: St. John's Wort 300 mg 3/d, Ginko 440 mg 2-3/d, GotuKola 450 mg 3/d, Ginsing 900 mg 3/d, Zinc 100 mg/d, Primrose oil 1000 mg2-3/d, Garlic 600 mg 3/d 011 hydroco 8/day Amitriptyline 2/day Xanflex5/day 012 016 Effexor xr 3 pills/day ginger rt Tab 1 pills/dayClonazepam 3 pills/day 017 no entry blank blank blank blank blank 018blank blank blank blank blank blank Patient GENERAL HEALTH -non-over/counter meds d 28 ID# meds? 001 1 amitriptyline, nasalcrom, E285 IU, Centrum (vit), carotenoid complex, B complex, glucosamine,magnesium/Ca, formula 4 enzymes lipids sterols, glaucoma opthalmics 0020 005 1 ?celebrex daily 006 1 Tiazac 240 mg, Ziac 5/6.25, Lipitor, 40Mg, Semprex-D 60 Mg, Atrovent Nasal Paxil 20 Mg, Synthroid, 50 Mg,Nexium, Centrum Silver, Vitamin C, 250 Mg, Spray, Vitamin E, 400iu,Caltrate & soy 007 1 Multivits occas 009 1 protein drinks 010 1Trazodone 150 mg/d, Zyrtec 10 mg/d, Valtrex 1 g/d, Wellbutrin sr 150 mg2/d, Albuterol 90 ug as needed 011 0 hydrocodone 2q4h(10/500 mg),Amitriptyline 200 mg qhs(200 mg qhs), Zanflex 5 tabs/d (4 mg), Orycontin2q8h(40 mg) 012 1 Trazodone (for sleep), calcium, multi vitamin 016 1Prilosec, Effexor xR, Trazodone, Clonazapam, Levsin, Relefen 017 1 multiVitamin-mineral: 1/day, Balanced B-100: 1/day, Vit E 400 IU: 1/day, VitC 250 mg: 1/day, Primadophilus-Bilidus 290 mg: 1/day 018 0 Blank

Example 2

This study will be performed to confirm the results of the trialdiscussed in Example 1 above. The study will be employ a randomized,double blind placebo controlled design in eighty to one hundredpatients. In the previous open label study of 12 patients, we tested thehypothesis that the fibromyalgia muscle pain and fatigue symptoms ofwomen, aged 40-60 with Fibromyalgia Syndrome (FMS), are caused byabnormally low circulating levels of testosterone. Further, we testedthe hypothesis that treatment of FMS patients with testosterone geldelivered transdermally will decrease the fibromyalgia-related pain andfatigue in these patients. The goals for this blinded efficacy study arevery similar. Specifically, the goals are to conduct a placebocontrolled study in FMS. for testosterone treatment forfibromyalgia-related pain and fatigue in FMS patients. Patients will betreated for 3 months, have a possible extension to 6 months, and willemploy symptomatic FMS efficacy endpoints, as in Example 1.

This study will be performed to confirm the dose of testosterone gelthat is appropriate for fibromyalgia subjects. The doses of testosteronegel used in the previous study approximate the blood levels expected fortestosterone replacement therapy in Fibromyalgia Syndrome (FMS)patients. The drug dose is based on the testosterone gel treatment inthe 12 patients, as discussed in Example 1. In our previous trial, FMSpatients were on the drug for 28 days and were tested for serumtestosterone levels pharmacokinetically over a 24 hr period both on day1 and at the end of treatment on day 28. The dose of drug used for thatstudy resulted in increased free testosterone serum levels that werelargely within the reference range.

The planned study will be performed to confirm the safety of the dose oftestosterone gel and the blood levels achieved with those doses. In thisstudy, safety checks will take place at weeks 4, 8, and 12 to monitorthe levels of testosterone in the blood. Dose reduction is an option ifneeded for safety purposes. A dose increase is an option, within thelimitations of safety, if needed for efficacy.

This study will be performed to assess testosterone blood levels due tothe testosterone treatment. The study will further continue to assessthe interpatient variation of testosterone blood levels achieved at thedoses of testosterone used and identify any remaining confoundingfactors when assessing testosterone levels.

The planned twelve-week research study will look at whether testosteronereplacement therapy can lessen the symptoms of fibromyalgia over 3-6months' time. Patients that are selected to participate in the studywill receive either testosterone gel or placebo gel and undergo anevaluation and blood tests once every four weeks. In addition, studyparticipants will complete patient questionnaires once every four weeks,take part in an exercise program and fill out daily medicine andexercise logs.

The target patients for this study will be 40-60 year old women who havebeen diagnosed as having fibromyalgia. The patients can be pre- orpost-menopausal, but should not be using hormone therapy (or will bewilling to cease such therapy). In addition, the patients should not useSt. John's Wort, antidepressants, ginger root (or will be willing tocease such use). In addition, in order to be selected for the study,patients will be required to exercise at least 20 minutes per day, fivedays a week, including stretching and aerobic exercise (walking,running, bicycling, climbing stairs, etc.).

The physical examination used to select participants for the study willinclude a “tender point” examination, which tests the intensity offibromyalgia-related muscle pain for each of the 18 commonly recognizedtender points that fibromyalgia patients are known to have. The tenderpoint examination will be repeated every four weeks.

Prospective study participants will be notified of the risks, and thelikelihood of their occurrence. The risks include increased libido(likely to occur); deepening of the voice, increased facial hair,temporal balding, acne, menstrual irregularities, lipid abnormalitiesand heart disease; liver toxicity (unlikely to occur); andhepatocellular carcinoma (rare occurrence). Patients whose blood levelsare maintained within the appropriate reference range for androgens areunlikely to have the toxicity risks associated with blood levels abovethe reference range.

This double blinded, placebo-controlled study with dose titration forsafety (see below) is designed to show in a controlled study thattestosterone replacement therapy results in decreasedfibromyalgia-related pain and fatigue in women with FibromyalgiaSyndrome (FMS). Female patients aged 40-60 who have been diagnosed withFMS and meet other eligibility criteria will be considered for thisPhase I/II study. A total of 80-100 outpatients will be enrolled, 40-50for each arm, drug versus placebo, and with randomization to initialtreatment versus placebo. Menstruating women will be started on drugwithin 10 days of their d1 of menses to initially control forreproductive endocrine state. A dose of testosterone gel, about 800 mggel at 0.8% w/w testosterone/gel (about 6.4 mg testosterone; at thecommonly accepted 10% bioavailability of drug in a gel =0.64 mgbioavailable testosterone, that is essentially equivalent to thatpreviously determined by us as appropriate for women (about 750 mg gelof 1% w/w testosterone/gel) versus placebo gel, will be applied to thesubject's abdominal skin at 0 hr on Day 1 (at 8:00 AM), and each morningthereafter once a day for 4 weeks. At 4 weeks, after serum testing fortestosterone levels, gel will be adjusted upward from 800 mg to 1200 mgfor patients whose levels remain in the low range (≦about 1.9 pg/mL freetestosterone, DSL test), and the gel will be adjusted downward fromabout 800 mg to about 400 mg for patients whose levels are high (>about3.3 pg/mL free testosterone, DSL test). Serum will be tested again at 8weeks and the dose readjusted similarly if necessary. Equivalent valueswill be used if Mayo Medical Labs testing is used.

This dose adjustment is important due to the individual variation wehave found between FMS subjects in response to testosterone therapy.This variation could be due to variation in catabolism rates, SHBGaffinity, etc. Our preliminary data (Example 1) allow us to predict thatwe can safely maintain subjects on a dose between 1-3 packets of about400 mg/packet about 0.8% testosterone gel per day. Testosterone levelswill be blinded to treatment staff and study investigators via use of a“Study Dosing Coordinator” who is distinct from the Study Coordinator.The three month open label (no placebo) continuation study may beextended to provide 6 month safety data for those patients originally ondrug, and to allow patients on placebo to go on drug for 3 months. Thedose will be adjusted similarly to how adjustments were made in thefirst 12 weeks, and we will use the same approach for testing.

To test for serum levels of testosterone (total and free), the studycoordinator will take blood samples (7 mL) by venipuncture prior to thestudy (for eligibility) and at 4, 8, and 12 weeks (and at 16, 20, 24weeks if the extension study is performed), between 8:00 and 9:00 AMprior to application of gel and after fasting since midnight the eveningbefore. Estradiol will be tested at d1, 12 wks (and 24 wk if theextension study is performed) to rule out conversion from testosteroneto estradiol in the circulation via aromatase. The limited dataavailable indicates that no such conversion takes place. Subjects willbe instructed to fast from midnight the evening before for all blooddraws.

For general health parameters, pre-study baseline samples andend-of-study samples will be collected at screening (Visit 1), 12 weeks(and 24 weeks for the extension study) for standard test panels (seeAppendix 1): cardiac health (Lipid Profile: total cholesterol, HDL, LDL,triglyceride), liver function (hepatic function panel: ALT, ALP, AST,albumin, TBiI, DBil), kidney function (BUN, creatinine) and CBC (toobtain hemoglobin levels since androgens are known to stimulate theproduction of red blood cells by enhancing erythropoietin production.FSH will be tested at the screening phase to discriminate pre- andpost-menopausal women and to allow for subgrouping in the analysis phase(high levels indicate the patient is postmenopausal). As notedpreviously, premenopausal women will be entered into drug treatmentwithin 10d of d1 of menses to initially control for endocrine state.

Testing for total testosterone serum levels will be conducted, as wellas for free testosterone serum levels. Testing for total testosterone ismost frequently done due to its lower cost, but free testosterone levelsallow for assessment of the biologically active compartment. Althoughfree testosterone levels have been found to correlate well with totaltestosterone levels, both are required for full analysis and both willbe tested here. Serum estradiol levels in these women were found to bewell within the reference range. Dihydrotestosterone (DHT) will not bemeasured since testosterone is converted to DHT within cells, andtestosterone is the norm for testing in women. DHT is dependent ontestosterone levels and can be assumed to correlate with testosteronelevels.

Sex hormone binding globulin (SHBG) reversibly binds testosteroneresulting in a bioavailable form (bioavailable testosterone is composedof free testosterone +testosterone loosely associated with globulin)versus a non-bioavailable form (SHBG bound). Testosterone and its moreform dihydrotestosterone have a higher affinity than estradiol for SHBG(Becker, p938). Androgens can decrease the binding affinity of SHBG forhormone, resulting in an increased bioavailability of both 17beta-hydroxyandrogens (testosterone and DHT) and estradiol (Becker,p837). Our testing of estradiol will allow us to determine howtestosterone therapy affects serum estradiol levels in our subjects.Estrogen administration is associated with a decrease in bioavailabletestosterone (Davis 1997). The patients studied here will not be onestrogen or any other hormone therapy at the time of testosteronetherapy.

Testosterone formulated in about an 0.8% (w/w) hydroalcoholic gel by anFDA licensed manufacturer, at about 800 mg gel/day (see details under“Dose of testosterone gel” below), will be prescribed for transdermaldelivery. The initial dose at about 800 mg will be adjusted to about 400mg or about 1200 mg as specified above based on free testosterone serumlevels tested at 4 week intervals. A packet-based or metered-dose methodcan be used to ensure delivery of an accurate quantity of gel. Otherdelivery systems can be contemplated as well, as long as the desiredblood levels are met. The gel will be applied to abdominal skin, abovethe waistline, for transdermal delivery by percutaneous absorption. Thegel dries in minutes and is colorless, comfortable and non-staining. Theform of testosterone used will be USP grade material of a compositionidentical in potency to a currently marketed transdermal gel which isFDA approved for men with hypogonadism.

The gel proposed for this study will be similar in composition to themarketed gel, but packaged and titrated for a female dosage regimen. Thegel will be compounded at an FDA-approved manufacturing site using GMPconditions appropriate for this stage of development, and will bemonitored for stability for the duration of the study. The gel will bemetered using a filled packet calibrated to deliver the required dose.The packets will be assembled in kits with the appropriate number ofpackets as determined by the Study Dose Coordinator, and with packageinsert instructions for the patient and scissors to snip the end of thepacket.

Testosterone is a schedule C-III controlled substance as defined by theAnabolic Steroids Control Act. The materials must be produced under aDEA license. The amounts dispensed are insufficient for abuse potential,and the samples will be itemized and accounted for. All unused materialscontaining the scheduled substance testosterone must be returned to theResearcher at the end of the study.

A transdermal route of delivery should provide benefits to the patientbeyond those found with oral delivery. The hormone is delivered over amore sustained time and at lower doses with improved bioavailability,thereby reducing the risk of hepatotoxic side effects. The injectable ortransdermal (gel, patch) forms of hormone largely circumvent thepossible hepatotoxicity issues associated with orally administeredhormones. Currently, the oral route of delivery is the predominant routeused by women on estrogen/progestin sex steroid hormone replacementtherapy. Delivery of testosterone via gel should be superior to oraldelivery. With regards to different vehicles for transdermal delivery, atestosterone patch is currently being studied in women for AIDS relatedwasting syndrome (Miller 1998, Javanbakht 2000). We prefer transdermaldelivery via gel, rather than patch, which will avoid the local skinirritation experienced by patients using the patch system since ˜30% ofpatients get contact dermatitis with the patch (P&G's Intrinsatestosterone patch for the subset of female sexual dysfunction (FSD)called Hypoactive Sexual Desire Disorder (HSDD).

Relatively high doses of androgens were given to patients in theearliest studies by others. The current thinking is to use these drugsfor amelioration of hormone deficiency at relatively low doses in anattempt to reconstitute normal levels. The planned study is in keepingwith this minimalist rationale. To determine the appropriate dose forour previous pharmacokinetic study using FMS patients (Example 1), weestimated the dose of testosterone gel based on the FDA approvedtestosterone gel product. Our pharmacokinetic methodology was similar tothat used for men in prior clinical trials.

All patients will be started with 2 packets of about 0.8% Testosteroneor Placebo gel per day for the first 4 weeks. Each packet has 400 mg of0.8% Testosterone gel (3.2 mg Testosterone, to deliver 10% or 320 μgbioavailable Testosterone) or 400 mg Placebo gel in it. The patient willbe shown where to rub the gel on her abdomen. After the 4 wk blood draw,any patient who tests >3.3 pg/mL for serum free Testosterone (HiT, DSLtest), decreases dose by 1 gel packet/d; any patient who tests ≦1.9pg/mL for serum free Testosterone (LoT, DSL test), increases dose by 1gel packet/d. Equivalent cut-off levels will be used if Mayo MedicalLabs testing is done instead. The decision to adjust the testosteronedose will be made again after the 8 wk blood draw. Our preliminary dataallow us to predict that we can safely maintain subjects on a dosebetween 1-3 packets of 400 mg 0.8% testosterone/packet per day. In orderto maintain the blind for the patient and the treating study staff,placebo patients will be split into three groups at week 4, with a thirdgiven 1 packet, a third maintained at 2 packets and a third given 3packets of Placebo.

The trial will be scheduled for 12 weeks to start, with a possibility ofextending the trial to a total of 24 weeks. If the study is able to beextended, all patients will receive open label testosterone starting at12 weeks, with the 12 week time point serving as the baseline for thePlacebo patients. Placebo patients will receive 2 packets ofTestosterone gel for the next 4 weeks. Possible dose adjustment forthese patients will take place after 16 weeks and again after 20 weeks,based on achieved free serum testosterone levels. Adjustments for allpatients will be made based on the same criteria as described above forweeks 4 and 8. For the subjects who were on drug during the first 12weeks, the additional 12 weeks of continuation will provide confirmationof efficacy and extended safety data. For the Placebo patients, thecontinuation study will ensure all patients in the study have a trial ofactive therapy.

The Study Coordinator or Study Resident will administer the FMSPreliminary Patient Questionnaire (FIG. 7). The Study Coordinator orStudy Resident will fill out the Eligibility Criteria Form (FIG. 8) andgive it to the physician. The Dosing Coordinator will fill out the DoseCoordinator Serum Testosterone Values Form (FIG. 9) and use it tocommunicate with the pharmacist and adjust the dose.

The physician will fill out a Physician Evaluation Form (FIG. 10) thatincludes the FMS Tender Point Exam prior to therapy to verify that thepatient fulfills the criteria for FMS, as defined by the AmericanCollege of Rheumatology, and to document the intensity offibromyalgia-related muscle pain for each of the 18 commonly recognizedtender points that patients with FMS are known to have (Okifuji, Turk1997). A dolorimeter will be used to quantify a subset of painfulpoints, similar to the tender point subsets used in our previous PKstudy: second costochondral (rib-cartilage) junction and trapezius atthe midpoint of the upper border, both for right and left sides, thus 4TP sites. The Physician's Form (FIG. 10) will be filled out at the endof the study at 12 weeks (and 24 weeks, if necessary), to allow acomparison of parameters before and after treatment.

The study coordinator (or study designated resident) will administer aPatient Questionnaire Form (FIGS. 12A-12D) to the study patient toassess their symptoms and level of fibromyalgia-related pain in asemi-quantitative manner, prior to therapy and at the end of the 12weeks of therapy (or at the time of early dropout from the study, and at24 weeks if the extension study is done) to allow a comparison ofparameters before and after treatment. Our Patient Questionnaire Form(FIGS. 12A-12D) is the same form as that used in our previouspharmacokinetic and efficacy study (Example 1) and is based on thevalidated Fibromyalgia Impact Questionnaire or FIQ (Burckhardt 1991).However, this form includes additional questions on gynecologicalhistory, exercise habits and use of analgesics, for example. Thequestionnaires and physical exam together include evaluation parametersthat are common to published and validated FMS patient questionnaires,such as fibromyalgia-related pain (e.g., tender point exam),sleeplessness, feeling refreshed, fatigue, headache and stiffness (Wolfe1990; Goldenberg 1996; Burckhardt 1991). A 100 mm visual analogue scale(VAS) will be used.

An initial summary assessment of the patient's general health viastandard physical exam will be noted on the Physician's General HealthForm (FIG. 11). For purposes of analysis, the study patient will beadministered the MOS SF-36 (Medical Outcomes Study Short Form with 36questions, FIGS. 13A-13F) to assess global health (Ware 1992, Picavet2004). This validated form was favorably reviewed by the FDA forevaluation of the general functional capacity to be used along with thepain instruments. It will be administered by the study coordinator priorto study for baseline, and at 12 wk (and 24 wk) at the end of study. TheSF-36 health survey form is a standard instrument for assessment ofglobal health.

The Mannerkorpi functional movement test will be administered by thestudy coordinator prior to study for baseline, and at 12 wk (and 24 wk)at the end of study. This instrument was chosen because of itsoutstanding ability to discriminate between FMS subjects and a controlgroup for range of motion and fibromyalgia-related pain perceived duringrange-of-motion testing using a VAS scale of 1-100 mm (Mannerkorpi 1999,P values at 0.0001 for pain perceived). This instrument has beenvalidated for FMS patients.

Patients will also be followed for changes in blood pressure and weight.At the end of the study, analyses will be conducted to show: 1) thepercentage of patients achieving >30% versus >50% improvement in theirpain scores by VAS, 2) the percentage of patients in the active arm whowould like to continue on with their same medication, 3) versus thepercentage of patients in the placebo arm who would like to continue onwith their same medication, 4) an analysis of use of pain relief networkpain medication in both arms, and 5) an analysis of dropouts due to lackof efficacy from both arms. The patients will be required to completethe examinations, questionnaires, blood draws, study gel logs (FIG. 16),and exercise logs (FIG. 17). The patients, if any occur, will also berequired to fill out an Adverse Event Form (FIG. 18). Each adverse eventwill be investigated by the study investigator.

Other forms that can be administered by the physician or DoseCoordinator for this study include the FMS Movement Test Form (FIGS.14A-14B).

1. A method of alleviating the symptoms of a condition which isassociated with deficient serum androgen levels in a female humanpatient comprising transdermally administering daily, to said patientsuffering from deficient serum androgen levels, a safe and effectiveamount of an androgen which is both effective for alleviating the femalepatient's condition associated with androgen deficiency and forconsistently raising the female patient's serum androgen levels to themiddle-upper female reference range, wherein the composition contains adaily unit dose of an androgen and is formulated to provide steady statetotal androgen serum levels without raising free androgen serum levelsor twenty-four hour free androgen AUC above the levels required for boththerapeutic efficacy and safety.
 2. The method of claim 1, wherein theandrogen is selected from the group consisting of testosterone,androstenedione, androstendiol, dehydroepiandrosterone, danazol,fluoxymesterone, oxandrolone, nandrolone decanoate, nandrolonephenpropionate, oxymethalone, stanozolol, methandrostenolone,testolactone, pregnenolone, dihydrotestosterone, methyltestosterone,androgen precursors, and testosterone esters.
 3. The method of claim 2,wherein the testosterone ester is selected from the group consisting oftestosterone enanthate and testosterone cypionate.
 4. The method ofclaim 2, wherein the androgen is testosterone.
 5. The method of claim 1,wherein the androgen is administered in a transdermal daily unit dose ofabout 1.0 mg to about 12.8 mg of the androgen, wherein theadministration results in steady state total androgen serum levelswithout raising free androgen serum levels or twenty-four hour freeandrogen AUC above the levels required for therapeutic efficacy andsafety.
 6. The method of claim 5, wherein the daily unit dose of theandrogen is from about 2.5 mg to about 10.0 mg.
 7. The method of claim6, wherein the daily unit dose of the androgen is from about 3.2 mg toabout 9.6 mg.
 8. The method of claim 6, wherein the daily unit dose ofthe androgen is from about 6.0 mg to about 8.0 mg.
 9. The method ofclaim 10, wherein the daily unit dose of the androgen is selected tomaintain steady state total androgen serum levels within a range ofbetween about 0.9 ng/mL to about 1.4 ng/mL for at least 24 hours afteradministration.
 10. The method of claim 10, wherein free androgen serumlevels and twenty-four hour free androgen AUC are not raised abovelevels required for therapeutic efficacy and safety.
 11. The method ofclaim 9, wherein the free androgen serum levels are raised to about 1.00pg/mL to about 3.30 pg/mL and the twenty-four hour free androgen AUClevels are raised to about 40.00 pg-h/mL to about 65.00 pg-h/mL.
 12. Themethod of claim 10, wherein the androgen is testosterone.
 13. The methodof claim 1, wherein the concentration of androgen is present in anamount of about 1% on a weight basis.
 14. The method of claim 1, whereinthe condition which is associated with deficient serum androgen levelsis selected from the group consisting of fibromyalgia, chronic fatiguesyndrome, and decreased sexual desire; and the safe female effectiveunit dose is an amount which will raise the female human patient'ssteady state serum androgen level without causing androgenic sideeffects.
 15. The method of claim 1, wherein administering thecomposition results in a low side effect profile.
 16. The method ofclaim 1, wherein the composition delivers a therapeutically effectiveand safe daily amount of the androgen to the patient's serum over each24 hour period to alleviate the patient's symptoms without causingandrogenic side effects.
 17. A method of determining the appropriateandrogen dosage for a female human subject comprising: diagnosing thefemale human subject as having fibromyalgia or chronic fatigue syndromeaccompanied by a free androgen blood level in the lower half of theappropriate reference range; starting transdermal androgen treatment ofthe subject based on a diagnosis of fibromyalgia or chronic fatiguesyndrome; measuring the free androgen level in the subject's blood aftera predetermined treatment time; wherein if the subject's free androgenlevel remains in the lower half of the appropriate reference range, theandrogen dosage is increased; if the subject's free androgen level iswithin the middle-to-upper end of the appropriate range, the androgendosage is maintained; or if the subject's free androgen level is inexcess of the standard error of the mean above the appropriate referencerange, the androgen dosage is decreased.
 18. The method of claim 16,wherein the androgen is selected from the group consisting oftestosterone, androstenedione, androstendiol, dehydroepiandrosterone,danazol, fluoxymesterone, oxandrolone, nandrolone decanoate, nandrolonephenpropionate, oxymethalone, stanozolol, methandrostenolone,testolactone, pregnenolone, dihydrotestosterone, methyltestosterone,androgen precursors, and testosterone esters.
 19. The method of claim17, wherein the testosterone ester is selected from the group consistingof testosterone enanthate and testosterone cypionate.
 20. The method ofclaim 17, wherein the androgen is testosterone.
 21. The method of claim16, wherein the androgen is administered in a transdermal daily unitdose of about 1.0 mg to about 12.8 mg of the androgen, wherein theadministration delivers a therapeutically effective daily amount of theandrogen to the patient's serum over each 24 hour period to alleviatethe patient's symptoms without causing androgenic side effects, andwherein the administration results in steady state total androgen serumlevels without raising free androgen serum levels or twenty-four hourfree androgen AUC above the levels required for therapeutic efficacy andsafety.